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Abstract:

The present invention relates to the field of recombinant protein
production in bacterial hosts. It further relates to extraction of
soluble, active recombinant protein from an insoluble fraction without
the use of denaturation and without the need for a refolding step. In
particular, the present invention relates to a production process for
obtaining high levels a soluble recombinant Type 1 interferon protein
from a bacterial host.

Claims:

1. A method for producing a recombinant Type 1 interferon protein, said
method comprising: expressing the recombinant interferon protein by
culturing a Pseudomonas or E. coli host cell containing an expression
construct comprising a coding sequence that has been optimized for
expression in the host cell; lysing the host cell; obtaining an insoluble
fraction and a soluble fraction from the lysis step; extracting the
insoluble fraction by subjecting it to non-denaturing extraction
conditions; and obtaining an extract pellet and an extract supernatant
from the insoluble fraction; wherein the recombinant protein in the
extract supernatant is present in soluble form, active form, or a
combination thereof, without being further subjected to a renaturing or
refolding step.

2. The method of claim 1, wherein the non-denaturing extraction
conditions comprise the presence of a mild detergent.

3. The method of claim 2, wherein the mild detergent is a Zwitterionic
detergent.

5. The method of claim 4, wherein the non-denaturing extraction
conditions comprise about 0.5% to about 2% Zwittergent 3-14.

6. The method of claim 2, wherein the non-denaturing extraction
conditions further comprise a chaotropic agent and a cosmotropic salt.

7. The method of claim 6, wherein the chaotropic agent is urea or
guanidinium hydrochloride, and wherein the cosmotropic salt is NaCl, KCl,
or (NH4)2SO.sub.4.

8. The method of claim 7, wherein the non-denaturing extraction
conditions comprise: about 0.5 to about 2% Zwittergent 3-14; about 0 to
about 2 M urea; about 0 to about 2 M NaCl; and wherein the pH is about
6.5 to about 8.5.

9. The method of claim 8, wherein the non-denaturing extraction
conditions comprise: about 1% Zwittergent 3-14; about 2 M urea; about 2 M
NaCl; and wherein the pH is about 8.2.

11. The method of claim 8, wherein the non-denaturing extraction
conditions additionally comprise about 1% to about 40% w/v solids.

12. The method of claim 1, wherein the recombinant Type 1 interferon
protein is an interferon-.beta., an interferon-.alpha., an
interferon-.kappa., or an interferon-.omega..

13. The method of claim 12, wherein the recombinant Type 1 interferon
protein is an interferon-.beta., and wherein said interferon-.beta. is
selected from the group consisting of: a human interferon-.beta. 1b and
human interferon-.beta. 1b C17S.

14. The method of claim 12, wherein the recombinant Type 1 interferon
protein is an interferon-.alpha., and wherein the interferon-.alpha. is
selected from the group consisting of: human interferon-.alpha. 2a and
human interferon-.alpha. 2b.

15. The method of claim 1, further comprising measuring the amount of
recombinant Type 1 interferon protein in the insoluble fraction and the
extract supernatant fractions, wherein the amount of recombinant
interferon protein detected in the extract supernatant fraction is about
10% to about 95% of the amount of the recombinant interferon protein
detected in the insoluble fraction.

16. The method of claim 1, further comprising measuring the activity of
the recombinant protein, wherein about 40% to about 100% of the
recombinant protein present in the extract supernatant is determined to
be active when compared with the total amount of recombinant protein
assayed.

17. The method of claim 1, wherein the recombinant protein in the extract
supernatant is present at a concentration of about 0.3 grams per liter to
about 10 grams per liter.

18. The method of claim 1, wherein the host cell is cultured in a volume
of about 1 to about 20 or more liters.

20. The method of claim 19, wherein the expression construct comprises a
lac promoter derivative and expression of the interferon is induced by
IPTG.

21. The method of claim 20, wherein the host cell is grown at a
temperature of about 25.degree. C. to about 33.degree. C., at a pH of
about 5.7 to about 6.5, and wherein the IPTG is added to a final
concentration of about 0.08 mM to about 0.4 mM, when the OD575 has
reached about 80 to about 160.

22. The method of claim 21, wherein the host cell is grown at a
temperature of about 32.degree. C., at a pH of about 5.7 to 6.25, and
wherein the IPTG is added to a final concentration of about 0.2 mM, when
the OD575 has reached about 120 to about 160.

25. The method of claim 13, wherein the human interferon-.beta. 1b or
human interferon-.beta. 1b C17S is expressed in the cytoplasm of the host
cell.

26. A method for extracting a recombinant Type 1 interferon protein,
wherein the recombinant interferon protein is present in an insoluble
fraction, said insoluble fraction produced after lysis of a Pseudomonas
or E. coli host cell expressing the recombinant interferon protein, said
method comprising: subjecting the insoluble fraction to non-denaturing
extraction conditions; and obtaining an extract pellet from the insoluble
fraction, said extract pellet comprising recombinant interferon protein;
wherein the recombinant interferon protein in the extract pellet is in
soluble form, active form, or a combination thereof, without being
subjected to a renaturing or refolding step.

27. A method for producing an insoluble fraction comprising a recombinant
Type 1 interferon protein, wherein the recombinant interferon protein is
expressed in a Pseudomonas or E. coli host cell from a nucleic acid
construct comprising a nucleic acid sequence that is operably linked to a
lac derivative promoter, said method comprising: growing the host cell at
a temperature of about 25.degree. C. to about 33.degree. C. and at a pH
of about 5.7 to about 6.5, to an OD600 of about 80 to about 160; and
inducing the host cell at a concentration of about 0.08 mM to about 0.4
mM IPTG; lysing the host cell and centrifuging it to produce the pellet
fraction; wherein soluble, active, or soluble and active recombinant
interferon protein can be obtained by extracting the pellet fraction
under non-denaturing conditions without a subsequent renaturing or
refolding step.

28. The method of claim 1, further comprising measuring the activity of
the recombinant protein, wherein about 75% to about 100% of the
recombinant protein present in the extract supernatant is determined to
be active when compared with the amount of active protein in a standard
sample, wherein the same amount of protein from each sample is used in
the assay.

Description:

CLAIM OF PRIORITY

[0001] This application claims priority under 35 U.S.C. §119(e) to
U.S. application Ser. No. 61/310,671 filed on Mar. 4, 2010. The contents
of U.S. application Ser. No. 61/310,671 are hereby incorporated by
reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Feb. 11, 2011, is
named 38194201.txt and is 9,237 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Many heterologous recombinant proteins are produced in a misfolded
insoluble form, called inclusion bodies, when expressed in bacterial
systems. In general, denaturing reagents must be used to solubilize the
recombinant protein in the inclusion bodies. The protein must then be
renatured, under conditions that have been optimized for the protein to
properly fold. Efforts expended on optimization, as well as the slow
refolding process and lowered process yields, add cost and time to the
production of a recombinant protein.

[0004] Interferons exhibit antiviral, antiproliferative, immunomodulatory,
and other activities. Several distinct types of human interferons,
including α, β, and γ, have been distinguished based on,
e.g., their anti-viral and anti-proliferative activities. Interferon
secretion is induced by signals, including viruses, double-stranded RNAs,
other polynucleotides, antigens, and mitogens. Interferon-β is an
example of a protein that has been expressed in recombinant form in
bacteria, where it is sequestered in inclusion bodies.

[0005] Human interferon-β 1b is a regulatory polypeptide having a
molecular weight of about 22 kDa and consisting of 165 amino acid
residues. It can be produced by many cells in the body, in particular
fibroblasts, in response to viral infection or exposure to other
biologics. It binds to a multimeric cell surface receptor. Productive
receptor binding results in a cascade of intracellular events leading to
the expression of interferon-β inducible genes and triggering
antiviral, antiproliferative and immunomodulatory activity.

[0006] Interferon-β 1b, specifically, Betaseron (h-IFN-β 1b
C17S), has been used to treat diseases including multiple sclerosis (MS),
hepatitis B and C infections, glioma, and melanoma. Interferon-β has
been demonstrated to reduce the number of attacks suffered by patients
with relapsing and remitting MS. Substantial amounts of interferon-β
1b are needed for therapeutic use. Recombinant interferon-β 1b has
been produced at low levels in mammalian cells, including human
fibroblasts and CHO cells. Animal cell expression is typically hindered
by technical difficulties including longer process time, easy
contamination of cultures, a requirement for maintaining stringent
culturing conditions, and the high cost of culture media. As the
glycoprotein component has been found to be generally unnecessary for the
activity of interferon β, research has turned to the expression of
the recombinant protein in the bacterial expression system, E. coli. As
noted, the inclusion bodies generated in E. coli must be solubilized by
denaturation, and the interferon-β refolded. Refolding, which is
slow, extends process time, adds cost, and lowers yield. To date, a
method for quickly and economically producing high levels of soluble
recombinant interferon-β in either mammalian or bacterial host
cells, without the need for denaturing and refolding steps, has not been
described.

SUMMARY OF THE INVENTION

[0007] The present invention relates to the expression of interferon in P.
fluorescens and development of a new method to extract active proteins
from the fermentation product using mild detergents and without the need
for a refolding process.

[0008] In particular, the present invention provides a method for
producing a recombinant Type 1 interferon protein, said method comprising
expressing the recombinant interferon protein by culturing a Pseudomonas
or E. coli host cell containing an expression construct comprising a
coding sequence that has been optimized for expression in the host cell,
lysing the host cell, obtaining an insoluble fraction and a soluble
fraction from the lysis step, extracting the insoluble fraction by
subjecting it to non-denaturing extraction conditions, and obtaining an
extract pellet and an extract supernatant from the insoluble fraction,
wherein the recombinant protein in the extract supernatant is present in
soluble form, active form, or a combination thereof, without being
further subjected to a renaturing or refolding step.

[0009] In embodiments, the non-denaturing extraction conditions comprise
the presence of a mild detergent. In certain embodiments, the mild
detergent is a Zwitterionic detergent. In specific embodiments, the
Zwitterionic detergent is Zwittergent 3-08, Zwittergent 3-10, Zwittergent
3-12, or Zwittergent 3-14. In embodiments, the non-denaturing extraction
conditions comprise about 0.5% to about 2% Zwittergent 3-14. In certain
embodiments, the mild detergent is not N-lauroyl-sarcosine (NLS).

[0010] In embodiments, the non-denaturing extraction conditions comprise
the presence of a mild detergent and further comprise a chaotropic agent
and a cosmotropic salt. In certain embodiments, the chaotropic agent is
urea or guanidinium hydrochloride, and the cosmotropic salt is NaCl, KCl,
or (NH4)2SO4. In specific embodiments, the non-denaturing extraction
conditions comprise about 0.5 to about 2% Zwittergent 3-14; about 0 to
about 2 M urea; about 0 to about 2 M NaCl; and the pH is about 6.5 to
about 8.5. In embodiments, the non-denaturing extraction conditions
comprise: 1% Zwittergent 3-14; 2 M urea; 2 M NaCl; and the pH is about
8.2. In other embodiments, the non-denaturing extraction conditions
additionally comprise about 1% to about 40% w/v solids. In certain
embodiments, the non-denaturing extraction conditions additionally
comprise about 5% w/v solids.

[0011] In embodiments, the recombinant Type 1 interferon protein is an
interferon-β, an interferon-α, an interferon-κ, an
interferon-τ, or an interferon-ω. In specific embodiments, the
recombinant Type 1 interferon protein is an interferon-β or an
interferon-α. In embodiments, the recombinant Type 1 interferon
protein is an interferon-β, and said interferon-β is selected
from the group consisting of: a human interferon-β 1b and human
interferon-β 1b C17S. In embodiments, wherein the recombinant Type 1
interferon is an interferon-α, the interferon-α is selected
from the group consisting of: human interferon-α 2a and human
interferon-α 2b.

[0012] In further embodiments the claimed method further comprises
measuring the amount of recombinant Type 1 interferon protein in the
insoluble fraction and the extract supernatant fractions, wherein the
amount of recombinant interferon protein detected in the extract
supernatant fraction is about 10% to about 95% of the amount of the
recombinant interferon protein detected in the insoluble fraction. In
other embodiments, the method further comprises measuring the activity of
the recombinant protein, wherein about 40% to about 100% of the
recombinant protein present in the extract supernatant is determined to
be active. In related embodiments, the recombinant protein is an
interferon-β, and the amount of active recombinant protein is
determined by Blue Sepharose affinity column chromatography, receptor
binding assay, antiviral activity assay, or cytopathic effect assay. In
other embodiments, the recombinant protein is an interferon-α, an
interferon-κ, or an interferon-ω, and the amount of active
recombinant protein is determined by Blue Sepharose affinity column
chromatography, receptor binding assay, antiviral activity assay, or
cytopathic effect assay.

[0013] The invention further includes methods for producing a recombinant
Type 1 interferon protein, said method comprising expressing the
recombinant interferon protein by culturing a Pseudomonas or E. coli host
cell containing an expression construct comprising a coding sequence that
has been optimized for expression in the host cell, lysing the host cell,
obtaining an insoluble fraction and a soluble fraction from the lysis
step, extracting the insoluble fraction by subjecting it to
non-denaturing extraction conditions, and obtaining an extract pellet and
an extract supernatant from the insoluble fraction, wherein the
recombinant protein in the extract supernatant is present in soluble
form, active form, or a combination thereof, without being further
subjected to a renaturing or refolding step, wherein the recombinant
protein is an interferon-β, and further wherein the non-denaturing
extraction conditions are optimized using the information in FIG. 4B.

[0014] In embodiments, the recombinant protein in the extract supernatant
is present at a concentration of about 0.3 grams per liter to about 10
grams per liter. In other embodiments, the host cell is cultured in a
volume of about 1 to about 20 or more liters. In specific embodiments,
the host cell is cultured in a volume of about 1 liter, about 2 liters,
about 3 liters, about 4 liters, about 5 liters, about 10 liters, about 15
liters, or about 20 liters.

[0015] In embodiments of the invention, the expression construct comprises
an inducible promoter. In specific embodiments, the expression construct
comprises a lac promoter derivative and expression of the interferon is
induced by IPTG.

[0016] In embodiments, the host cell is grown at a temperature of about
25° C. to about 33° C., at a pH of about 5.7 to about 6.5,
and the IPTG is added to a final concentration of about 0.08 mM to about
0.4 mM, when the OD575 has reached about 80 to about 160. In specific
embodiments, the host cell is grown at a temperature of about 32°
C., at a pH of about 5.7 to 6.25, and the IPTG is added to a final
concentration of about 0.2 mM, when the OD575 has reached about 120 to
about 160.

[0017] In embodiments of the invention, the expression construct comprises
a high activity ribosome binding site. In certain embodiments, the host
cell is a lon hslUV protease deletion strain. In other embodiments, the
Type 1 interferon is expressed in the cytoplasm of the host cell. In
related embodiments, the Type 1 interferon is human interferon-β 1b
or human interferon-β 1b C17S, and is expressed in the cytoplasm of
the host cell.

[0018] The invention also provides methods for extracting a recombinant
Type 1 interferon protein, wherein the recombinant interferon protein is
present in an insoluble fraction, said insoluble fraction produced after
lysis of a Pseudomonas or E. coli host cell expressing the recombinant
interferon protein, said method comprising subjecting the insoluble
fraction to non-denaturing extraction conditions, and obtaining an
extract pellet from the insoluble fraction, said extract pellet
comprising recombinant interferon protein, wherein the recombinant
interferon protein in the extract pellet is in soluble form, active form,
or a combination thereof, without being subjected to a renaturing or
refolding step.

[0019] In embodiments, the recombinant Type 1 interferon protein extracted
is an interferon-β, an interferon-α, an interferon-κ, an
interferon-τ, or an interferon-ω. In certain embodiments, the
recombinant Type 1 interferon protein is an interferon-β or an
interferon-α. In embodiments, the recombinant Type 1 interferon
protein is an interferon-β, and said interferon-β is selected
from the group consisting of: a human interferon-β 1b and human
interferon-β 1b C17S. In other embodiments, the interferon-α
is selected from the group consisting of: human interferon-α 2a and
human interferon-α 2b.

[0020] The invention additionally provides a method for producing an
insoluble fraction comprising a recombinant Type 1 interferon protein,
wherein the recombinant interferon protein is expressed in a Pseudomonas
or E. coli host cell from a nucleic acid construct comprising a nucleic
acid sequence that is operably linked to a lac derivative promoter, said
method comprising growing the host cell at a temperature of about
25° C. to about 33° C. and at a pH of about 5.7 to about
6.5, to an OD600 of about 80 to about 160, and inducing the host cell at
a concentration of about 0.08 mM to about 0.4 mM IPTG, lysing the host
cell and centrifuging it to produce the pellet fraction, wherein soluble,
active, or soluble and active recombinant interferon protein can be
obtained by extracting the pellet fraction under non-denaturing
conditions without a subsequent renaturing or refolding step.

[0021] In embodiments, the recombinant Type 1 interferon protein comprised
by the insoluble fraction is an interferon-β, an interferon-α,
an interferon-κ, or an interferon-ω. In specific embodiments,
the recombinant Type 1 interferon protein is an interferon-β or an
interferon-α. In embodiments, wherein recombinant Type 1 interferon
protein is an interferon-β, the interferon-β is selected from
the group consisting of: a human interferon-β 1b and human
interferon-β 1b C17S. In embodiments, wherein the Type 1 interferon
protein is an interferon-α, the interferon-α is selected from
the group consisting of: human interferon-α 2a and human
interferon-α 2b. In embodiments, in the method for producing an
insoluble fraction comprising a recombinant Type 1 interferon protein,
the temperature at which the host cell is grown is about 32° C.,
and the IPTG concentration is about 0.2 mM.

INCORPORATION BY REFERENCE

[0022] All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent application
was specifically and individually indicated to be incorporated by
reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained by
reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention are
utilized, and in the accompanying drawings.

[0041] The present invention relates to a method for producing large
amounts of soluble recombinant interferon protein in a Pseudomonas
expression system. In particular, this method eliminates the need for the
denaturing step and subsequent renaturing/refolding step typically
required.

[0042] Production of recombinant interferon-β in bacterial expression
systems has been hampered by sequestration of the protein in insoluble
inclusion bodies. Solubilization of the inclusion bodies requires
denaturation, which in turn necessitates the use of a refolding step that
is costly, time-consuming, and decreases protein yield. The present
invention circumvents the need for a refolding step by providing methods
for producing and solubilizing interferon without recourse to
denaturation.

[0043] Methods for producing a recombinant interferon protein that is
soluble, active, or both, in a bacterial expression system, without
subjecting the protein to a denaturing step, are provided. In particular,
a non-denaturing extraction process that results in soluble interferon
protein is described. Interferons expressed in bacterial expression
systems are generally localized to an insoluble fraction. In the
extraction process of the present invention, this insoluble fraction is
subjected to extraction conditions that include non-denaturing
concentrations of mild detergents and produce soluble protein.

[0044] Also provided by the present invention are methods for producing a
recombinant interferon protein wherein growth conditions for the
Pseudomonas host cell are optimized to maximize yields of the soluble
recombinant interferon protein, particularly when the extraction method
of the invention is used. Studies of the effect of E. coli growth
conditions on soluble protein production have been reported. The
solubility of a given protein when expressed in Pseudomonas can be
different from that in E. coli. This is illustrated in, e.g., U.S. Pat.
App. Pub. No. 2006/0040352, "Expression of Mammalian Proteins in
Pseudomonas Fluorescens," which shows side-by-side comparisons of the
soluble amounts of several proteins produced using E. coli or P.
fluorescens as the host. Furthermore, there is substantial variation
among the solubilities of different proteins even in the same host, as
solubility is influenced strongly by protein structure, e.g., amino acid
sequence. Previously reported attempts at producing IFN-β in E. coli
resulted in protein that required refolding. See, e.g., Russell-Harde,
1995, "The Use of Zwittergent 3-14 in the Purification of Recombinant
Human Interferon-β Ser17 (Betaseron) et al., J. Interferon and
Cytokine Res. 15:31-37, and Ghane, et al., 2008, "Over Expression of
Biologically Active Interferon Beta Using Synthetic Gene in E. coli," J.
of Sciences, Islamic Republic of Iran 19(3):203-209, both incorporated
herein by reference.

[0045] The methods further provide optimized growth conditions including
growth temperature, OD at time of promoter induction, inducer
concentration, and pH. Extraction conditions provided include detergent
type and concentration, chaotropic agent, cosmotropic salt, and pH.
Specific values as well as optimal parameter ranges are provided. Also
provided are methods for optimizing extraction conditions using the
provided ranges.

Bacterial Growth Conditions

[0046] In embodiments of the present invention, the bacterial growth
conditions are optimized to increase the amount of soluble interferon
protein obtained using the extraction methods as provided herein. Use of
the growth conditions of the present invention with other extraction
conditions, e.g., other methods described and used in the art, is also
contemplated.

[0047] Optimal growth conditions particularly useful in conjunction with
the extraction methods of the invention comprise: a temperature of about
25° C. to about 33° C.; a pH of about 5.7 to about 6.5, and
induction with about 0.08 mM to about 0.4 mM IPTG when the culture has
reached an OD575 of about 80 to about 160.

[0048] The pH of the culture can be maintained using pH buffers and
methods known to those of skill in the art. Control of pH during
culturing also can be achieved using aqueous ammonia. In embodiments, the
pH of the culture is about 5.7 to about 6.5. In certain embodiments, the
pH is 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4. or 6.5. In other
embodiments, the pH is 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2,
6.1 to 6.3, or 6.2 to 6.5. In yet other embodiments, the pH is 5.7 to
6.0, 5.8 to 6.1, 5.9 to 6.2, 6.0 to 6.3, 6.1 to 6.4, or 6.2 to 6.5. In
certain embodiments, the pH is about 5.7 to about 6.25.

[0049] In embodiments, the growth temperature is maintained at about
25° C. to about 33° C. In certain embodiments, the growth
temperature is about 25° C., about 26° C., about 27°
C., about 28° C., about 29° C., about 30° C., about
31° C., about 32° C., or about 33° C. In other
embodiments, the growth temperature is maintained at about 25° C.
to about 27° C., about 25° C. to about 28° C., about
25° C. to about 29° C., about 25° C. to about
30° C., about 25° C. to about 31° C., about
25° C. to about 32° C., about 25° C. to about
33° C., about 26° C. to about 28° C., about
26° C. to about 29° C., about 26° C. to about
30° C., about 26° C. to about 31° C., about
26° C. to about 32° C., about 27° C. to about
29° C., about 27° C. to about 30° C., about
27° C. to about 31° C., about 27° C. to about
32° C., about 26° C. to about 33° C., about
28° C. to about 30° C., about 28° C. to about
31° C., about 28° C. to about 32° C., about
29° C. to about 31° C., about 29° C. to about
32° C., about 29° C. to about 33° C., about
30° C. to about 32° C., about 30° C. to about
33° C., about 31° C. to about 33° C., about
31° C. to about 32° C., about 30° C. to about
33° C., or about 32° C. to about 33° C.

Induction

[0050] As described elsewhere herein, inducible promoters can be used in
the expression construct to control expression of the recombinant
interferon gene. In the case of the lac promoter derivatives or family
members, e.g., the tac promoter, the effector compound is an inducer,
such as a gratuitous inducer like IPTG
(isopropyl-β-D-1-thiogalactopyranoside, also called
"isopropylthiogalactoside"). In embodiments, a lac promoter derivative is
used, and interferon expression is induced by the addition of IPTG to a
final concentration of about 0.08 mM to about 0.4 mM, when the cell
density has reached a level identified by an OD575 of about 80 to
about 160.

[0051] In embodiments, the OD575 at the time of culture induction
about 80, about 90, about 100, about 110, about 120, about 130, about
140, about 150, about 160, about 170 or about 180. In other embodiments,
the OD575 is about 80 to about 100, about 100 to about 120, about
120 to about 140, about 140 to about 160. In other embodiments, the
OD575 is about 80 to about 120, about 100 to about 140, or about 120
to about 160. In other embodiments, the OD575 is about 80 to about
140, or about 100 to 160. The cell density can be measured by other
methods and expressed in other units, e.g., in cells per unit volume. For
example, an OD575 of about 80 to about 160 of a Pseudomonas
fluorescens culture is equivalent to approximately 8×1010 to
about 1.6×1011 colony forming units per mL or 35 to 70 g/L dry
cell weight. In embodiments, the cell density at the time of culture
induction is equivalent to the cell density as specified herein by the
absorbance at OD575, regardless of the method used for determining
cell density or the units of measurement. One of skill in the art will
know how to make the appropriate conversion for any cell culture.

[0052] In embodiments, the final IPTG concentration of the culture is
about 0.08 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, or about 0.4 mM.
In other embodiments, the final IPTG concentration of the culture is
about 0.08 mM to about 0.1 mM, about 0.1 mM to about 0.2 mM, about 0.2 mM
to about 0.3 mM, about 0.3 mM to about 0.4 mM, about 0.2 mM to about 0.4
mM, or about 0.08 to about 0.2 mM.

[0053] In embodiments, the interferon is expressed by induction of a lac
promoter or derivative using IPTG, lactose or allolactose, by methods
known in the art and described in the literature, e.g., in U.S. Pat. No.
7,759,109, "High density growth of T7 expression strains with
auto-induction option," incorporated herein by reference in its entirety.

[0054] In embodiments wherein a non-lac type promoter is used, as
described herein and in the literature, other inducers or effectors can
be used.

[0055] After induction is started, cultures are grown for a period of
time, typically about 24 hours, during which time the recombinant
interferon protein is expressed. Cell cultures can be concentrated by
centrifugation, and the culture pellet resuspended in a buffer or
solution appropriate for the subsequent lysis procedure.

[0056] In embodiments, cells are disrupted using equipment for high
pressure mechanical cell disruption (which are available commercially,
e.g., Microfluidics Microfluidizer, Constant Cell Disruptor, Niro-Soavi
homogenizer or APV-Gaulin homogenizer). Any appropriate method known in
the art for lysing cells can be used to release the insoluble fraction.
For example, in embodiments, chemical and/or enzymatic cell lysis
reagents, such as cell-wall lytic enzyme and EDTA, can be used. Use of
frozen or previously stored cultures is also contemplated in the methods
of the invention. Cultures can be OD-normalized prior to lysis.

[0057] Centrifugation is performed using any appropriate equipment and
method. Centrifugation of cell culture or lysate for the purposes of
separating a soluble fraction from an insoluble fraction is well-known in
the art. For example, lysed cells can be centrifuged at 20,800×g
for 20 minutes (at 4° C.), and the supernatants removed using
manual or automated liquid handling. The pellet (insoluble) fraction is
resuspended in a buffered solution, e.g., phosphate buffered saline
(PBS), pH 7.4. Resuspension can be carried out using, e.g., equipment
such as impellers connected to an overhead mixer, magnetic stir-bars,
rocking shakers, etc.

[0058] A "soluble fraction," i.e., the soluble supernatant obtained after
centrifugation of a lysate, and an "insoluble fraction," i.e., the pellet
obtained after centrifugation of a lysate, result from lysing and
centrifuging the cultures. These two fractions also can be referred to as
a "first soluble fraction" and a "first insoluble fraction,"
respectively.

[0059] It is possible to obtain soluble IFN-β using extraction
methods according to the invention, from expression cultures prepared by
growing cultures under conditions in which the pH and the induction OD
are not tightly controlled (see, e.g., Example 2). Optimization of the
growth conditions as described herein results in substantially increased
production of soluble IFN-β.

Non-Denaturing Extraction Process

[0060] It has been discovered that high levels of soluble interferon
protein can be obtained from the insoluble fraction, using non-denaturing
extraction methods of the present invention.

[0061] Non-denaturing extraction conditions identified as particularly
useful for producing high levels of soluble recombinant interferon
protein comprise: a mild detergent at a non-denaturing concentration,
e.g., Zwittergent 3-14 (0.5 to 2% w/v); a chaotropic agent, e.g., urea
(0-2M), and a cosmotropic salt, e.g., NaCl (0-2M), at a buffer pH of 6.5
to 8.5 and a solids concentration of 5-20% w/v.

[0062] After obtaining the soluble fraction and insoluble fraction, as
described above, the soluble recombinant interferon protein is extracted
from the insoluble fraction by incubating the resuspended insoluble
fraction under the non-denaturing extraction conditions described herein.
After incubation, the extracted mixture is centrifuged to produce an
"extract supernatant" (the soluble supernatant fraction obtained after
extraction containing solubilized recombinant protein) and an "extract
pellet" (the insoluble pellet fraction obtained after extraction). These
fractions can also be referred to as the "second soluble fraction" and
the "second insoluble fraction."

Extraction Conditions

[0063] Many different parameters for the extraction conditions were
evaluated for their effect on the amount of soluble protein observed in
the extract supernatant, as described in Example 3 herein. It was found
that soluble interferon protein was observed when the extraction
conditions comprised any of a number of different detergents, at varying
concentrations, as well as when other parameters were varied. However,
certain parameters had a particularly striking effect on the amount of
soluble protein produced.

[0064] Specifically, extraction conditions comprising Zwitterionic
detergents (Zwittergents) gave the best soluble protein yields. In
particular, use of the Zwitterionic detergents, Zwittergent 3-08,
Zwittergent 3-10, Zwittergent 3-12, and especially Zwittergent 3-14,
resulted in the highest yields. N-Lauroylsarcosine (NLS) gave a notably
high yield, however the soluble protein obtained was found to be inactive
based on an affinity assay (Sepharose blue affinity column binding).
Therefore, the term "mild detergents" as used herein is intended not to
include N-lauroylsarcosine.

[0065] The detergents were tested at non-denaturing concentrations. It was
found that a concentration of Zwittergent 3-14
(3-(N,N-dimethylmyristylammonio) propanesulfonate) of at least 0.5%
(w/v), and preferably 1%, well above its critical micelle concentration
(which is 0.01%) provides the most efficient extraction of soluble
interferon protein.

[0066] Therefore, use of non-denaturing concentrations of mild detergents,
particularly Zwitterionic detergents, more particularly Zwittergent 3-08,
Zwittergent 3-10, Zwittergent 3-12, and Zwittergent 3-14, more
particularly Zwittergent 3-14, and not NLS, is contemplated for use in
the extraction conditions of the invention.

[0067] In other embodiments of the invention, the non-denaturing
extraction conditions comprise a concentration of about 0.5% to about 2%
(w/v) Zwittergent 3-14. In embodiments, the w/v concentration of
Zwittergent 3-14 is about 0.5% to about 1%, about 1% to about 1.5%, about
1.5% to about 2%, or about 1% to about 2%. In certain embodiments, the
w/v concentration of Zwittergent 3-14 is about 0.5%, about 0.6%, about
0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about
1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about
1.9%, or about 2.0%.

[0068] In other embodiments of the invention, non-denaturing extraction
conditions comprise a concentration of about 0.5% to about 2% (w/v)
Zwittergent 3-08, Zwittergent 3-10, or Zwittergent 3-12. In embodiments,
the w/v concentration of Zwittergent 3-08, Zwittergent 3-10, or
Zwittergent 3-12 is about 0.5% to about 1%, about 1% to about 1.5%, about
1.5% to about 2%, or about 1% to about 2%. In certain embodiments, the
w/v concentration of Zwittergent 3-08, Zwittergent 3-10, or Zwittergent
3-12 is about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about
1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%.

[0069] A mild detergent does not cause protein unfolding at low levels,
e.g., 2% or less. For example, SDS and NLS are typically considered
stronger detergents, as they can denature proteins at these levels. A
non-denaturing concentration indicates a concentration of a reagent at
which proteins are not denatured. Proteins that are not denatured during
processing do not require refolding.

[0070] In embodiments, non-denaturing extraction conditions of the present
invention comprise about 0.5 to about 2% Zwittergent 3-14; about 0 to
about 2 M urea; about 0 to about 2 M NaCl; and wherein the pH is about
6.5 to about 8.5.

[0071] The following table lists examples of common detergents, including
ionic, non-ionic, and zwitter-ionic detergents, and their properties. An
important characteristic of a detergent is its critical micelle
concentration (CMC), which relates to its protein solubilization
capability as well as the ease of subsequent removal of detergents from
protein solutions.

[0072] It was further observed that when the non-denaturing extraction
conditions comprised the combination of a chaotropic agent, urea, a
cosmotropic salt, NaCl, Zwittergent 3-14, and an appropriate buffer
range, the extraction yield was increased several-fold compared to the
use of Zwittergent 3-14 alone (see Example 3).

[0073] Chaotropic agents disrupt the 3-dimensional structure of a protein
or nucleic acid, causing denaturation. In embodiments, the non-denaturing
extraction conditions comprise low, non-denaturing concentrations of
chaotropic agents, e.g., urea or guanidinium hydrochloride. In
embodiments, the non-denaturing extraction conditions comprise urea at a
concentration of about 0.5M to about 2M or higher. We observed that 6M
urea denatures IFN-β. Typically, non-denaturing concentrations of
urea or guanidinium hydrochloride are below 3M. In embodiments, the
non-denaturing extraction conditions comprise urea at a concentration of
about 0.5 to about 1M, about 1 to about 1.5M, about 1.5 to about 2M,
about 1 to about 2M, about 0.5M, about 0.6M, about 0.7M, about 0.8M,
about 0.9M, about 1.0M, about 1.1M, about 1.2M, about 1.3M, about 1.4M,
about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, or about
2.0M. In other embodiments, the extraction conditions comprise
guanidinium hydrochloride at a concentration of 0.5 to 2M. In
embodiments, extraction conditions comprise guanidinium hydrochloride at
a concentration of 0.5 to 1M, 1 to 1.5M, 1.5 to 2M, 1 to 2M, 0.5M, about
0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M, about
1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about
1.8M, about 1.9M, or about 2.0M.

[0074] Cosmotropic salts contribute to the stability and structure of
water-water interactions. Cosmotropes cause water molecules to favorably
interact, which also stabilizes intermolecular interactions in
macromolecules such as proteins. Any such appropriate agent, as known in
the art, can be used in the extraction conditions of the present
invention. In embodiments, the non-denaturing extraction conditions
comprise a cosmotropic salt selected from NaCl, KCl, and
(NH4)2SO4. In certain embodiments, NaCl is present at a
concentration of about 0.15M to about 2M. In embodiments, NaCl is present
in the extraction conditions at a concentration of about 0.15 to about
0.5M, about 0.5 to about 0.75M, about 0.75M to about 1M, about 1M to
about 1.25M, about 1.25M to about 1.5M, about 1.5M to about 1.75M, about
1.75M to about 2M, about 0.15M to about 1M, about 1M to about 1.5M, about
1.5M to about 2M, about 1M to about 2M, about 0.15M, about 0.25M, about
0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about
1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about
1.7M, about 1.8M, about 1.85M, about 1.9M, or about 2.0M.

[0075] In other embodiments, KCl is present in the non-denaturing
extraction conditions at a concentration of about 0.15 to about 0.5M,
about 0.5 to about 0.75M, about 0.75M to about 1M, about 1M to about
1.25M, about 1.25M to about 1.5M, about 1.5M to about 1.75M, about 1.75M
to about 2M, about 0.15M to about 1M, about 1M to about 1.5M, about 1.5M
to about 2M, about 1M to about 2M, about 0.15M, about 0.25M, about 0.5M,
about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M,
about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M,
about 1.8M, about 1.85M, about 1.9M, or about 2.0M.

[0076] In other embodiments, (NH4)2SO4 is present in the
non-denaturing extraction conditions at a concentration of about 0.15 to
about 0.5M, about 0.5 to about 0.75M, about 0.75M to about 1M, about 1M
to about 1.25M, about 1.25M to about 1.5M, about 1.5M to about 1.75M,
about 1.75M to about 2M, about 0.15M to about 1M, about 1M to about 1.5M,
about 1.5M to about 2M, about 1M to about 2M, about 0.15M, about 0.25M,
about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M,
about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M,
about 1.7M, about 1.8M, about 1.85M, about 1.9M, or about 2.0M.

[0077] The extraction conditions were found to be most effective when the
pH was maintained within a range of 6.5 to 8.5. Useful pH buffers are
those recommended in standard protein purification texts (e.g., Protein
Purification: Principles and Practice, by Robert Scopes (Springer, 1993)
can be used here, including Tris, Bis-Tris, phosphate, citrate, acetate,
glycine, diethanolamine, 2-amino-2-methyl-1,3-propanediol,
triethanolamine, imidazole, histidine, pyridine, etc. Many buffers have
been described in the literature and are commercially available. In
embodiments, the pH of the non-denaturing extraction conditions is about
6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,
about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.8, about
7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5.
In other embodiments, the pH is about 6.5 to about 6.8, about 6.6 to
about 6.9, about 6.7 to about 7.0, about 6.8 to about 7.1, about 6.9 to
about 7.2, about 7.0 to about 7.3, about 7.1 to about 7.4, about 7.2 to
about 7.5, about 7.3 to about 7.6, about 7.4 to about 7.7, about 7.5 to
about 7.8, about 7.6 to about 7.9, about 7.8 to about 8.1, about 7.9 to
about 8.2, about 8.0 to about 8.3, about 8.1 to about 8.4 or about 8.2 to
about 8.5. In other embodiments, the pH is about 6.5 to about 7.0, about
7.0 to about 7.5, or about 7.5 to about 8.0.

[0078] The solids concentration in the non-denaturing extraction
conditions was also varied. This parameter represents the amount of solid
material in the extract incubation mixture. Solids concentration can be
determined by weighing the wet pellet (i.e., the insoluble fraction), and
comparing this weight with the total weight of the extraction mixture.
Specific solids concentrations are achieved by concentrating or diluting
the insoluble fraction. High extraction yields were observed across a
range of solids concentrations of 5% to 40% (w/v). In embodiments of the
invention, the solids in the non-denaturing extraction conditions are
present at a w/v concentration of about 5%, about 7.5%, about 10%, about
12.5%, about 15%, about 17.5%, about 20%, about 22.5%, about 25%, about
27.5%, about 30%, about 32.5%, about 35%, about 37.5%, or about 40%. In
other embodiments of the invention, the solids in the non-denaturing
extraction conditions are present at a w/v concentration of about 5% to
about 7.5%, about 7.5% to about 10%, about 10% to about 12.5%, about
12.5% to about 15%, about 15% to about 17.5%, about 17.5% to about 20%,
about 20% to about 22.5%, about 22.5% to about 25%, about 25% to about
27.5%, about 27.5% to about 30%, about 30% to about 32.5%, about 32.5% to
about 35%, about 35% to about 37.5%, about 37.5% to about 40%, about 5%
to about 10%, about 10% to about 15%, about 15% to about 20%, about 20%
to about 25%, about 25% to about 30%, about 35% to about 40%, about 5% to
about 15%, about 5% to about 25%, about 5% to about 30%, about 5% to
about 35%, about 10% to about 20%, about 20% to about 30%, about 30% to
about 40%, about 5% to about 20%, or about 20% to about 40%.

[0079] In embodiments, the extraction methods of the invention are
combined with simultaneous enrichment techniques such as adsorption to
further enhance protein yield.

[0080] The solubilized protein can be isolated or purified from other
protein and cellular debris by, for example, centrifugation and/or
chromatography such as size exclusion, anion or cation exchange,
hydrophobic interaction, or affinity chromatography.

Interferons

[0081] Human interferons have been classified into three major types.
Interferon type I: Type I IFNs bind to a specific cell surface receptor
complex known as the IFN-α receptor (IFNAR) that consists of IFNAR1
and IFNAR2 chains. Human type I interferons include are IFN-α,
IFN-β, IFN-κ, and IFN-ω and IFN-ε. Interferon
type II: Type II IFNs (human IFN-γ) binds to IFNGR. Interferon type
III: type III interferons signal through a receptor complex consisting of
IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12).

[0082] Human Type I interferon appears to bind to two-receptor subunits,
IFNAR-1 and -2, which are widely distributed on the cell surface of
various cell types. Ligand involvement leads to the induction of the
phosphorylation of tyrosine kinases TYK2 and JAK-1, which are coupled to
IFNAR-1 and -2 respectively. Once phosphorylated, STAT proteins are
released from the receptor and form homodimers as well as heterodimers.
Once released, the dimers of STAT associate with interferon Responsive
Factor 9 (IRF-9), a DNA binding protein, forming a complex called
IFN-stimulated gene factor-3 (ISGF-3), that migrates into the nucleus.
Next, the ISGF-3 complex binds to a DNA element existing in the upstream
of all IFN inducible genes. Type 1 interferons are described extensively
in the literature, e.g., in U.S. Pat. No. 7,625,555, "Recombinant human
interferon-like proteins, incorporated herein by reference."

[0083] Type 1 IFNs have substantial structural similarity, as evidenced by
their sequences and their shared receptor binding capacity. According to
Kontsek, P., 1994, "Human type I interferons: structure and function,"
Acta Virol. 38(6):345-60, incorporated by reference herein, human type I
interferons (13 had been reported at the time) have a 20% overall
sequence homology, which determines identical secondary and tertiary
folding of polypeptides. Further, Kontsek reports that three-dimensional
models suggest that the globular structure of type I IFNs consists of a
bundle of 5 α-helices, which might form two polypeptide domains.
Disulfide bond Cys 29-Cys 139 stabilizes both domains in a bioactive
configuration. Two conservative hydrophilic regions associated with the
amino acids (aa) 30-41 and 120-145 are thought to constitute the basic
framework of receptor recognition site in type I IFNs, and the different
spectra of biological effects among human type I IFNs are speculated to
be due to subtle sequential heterogeneity in the segments aa 30-41 and
120-145, and the variable hydrophilic aa regions 23-26, 68-85 and
112-121. A later report by Oritani, et al., 2001, "Type I interferons and
limitin: a comparison of structures, receptors, and functions," Cytokine
Growth Factor Rev 12(4):337-48, incorporated by reference herein,
describes family members IFN-α, IFN-β, IFN-pi, and IFN-tau.
The paper also reports that IFN-α and IFN-β have a globular
structure composed of five α-helices, and discusses comparative
sequence analyses, a prototypic three-dimensional structure, analysis
with monoclonal antibodies, and construction of hybrid molecules and site
directed mutagenesis.

[0084] Production of any Type 1 interferon protein using the methods of
the present invention is contemplated. Type 1 interferon proteins that
can be produced using the methods of the invention, include, but not
limited to, human IFN-α 2a and 2b, human IFN-β 1b, human
IFN-κ (e.g., NM--020124, AAK63835, and described by LaFleur,
et al., 2001, "Interferon-kappa, a novel type I interferon expressed in
human keratinocytes," J. Biol. Chem. 276 (43), 39765-39771, incorporated
herein by reference), and human IFN-ω (e.g., NM--002177,
NP--002168, and described in U.S. Pat. No. 7,470,675, "Methods for
treating cancer using interferon-ω-expressing polynucleotides,"
incorporated by reference herein in its entirety). Production of
IFN-τ using the methods of the invention is also contemplated. Amino
acid and nucleic acid sequences are publicly available, e.g., from
GenBank.

[0086] IFN-β (IFNB1, or IFN-β 1b) is the main 0 subtype (see,
e.g., GenBank NP002167.1, which provides the mature peptide sequence).
Betaseron is an analogue of human IFN-β in which serine was
genetically engineered to substitute for cysteine at position 17, is
known as IFN-β 1b C17S (described in U.S. Pat. No. 4,588,585, "Human
recombinant cysteine depleted interferon-β muteins," incorporated
herein by reference). The molecule is a small polypeptide of 165 amino
acids with a single disulphide bond, and is produced as a
non-glycosylated protein.

[0087] IFN-τ is described, and sequences of IFN-τ disclosed, e.g.,
in U.S. Pat. No. 7,214,367, "Orally-administered interferon-tau
compositions and methods," incorporated herein by reference in its
entirety.

[0088] A number of Type 1 IFNs have been approved by the FDA for use in
treating disease in humans. The following table lists examples of Type 1
interferon drugs. In embodiments of the invention, any of these drugs are
produced using the methods as claimed or described herein.

[0089] In embodiments, variants and modifications of Type 1 interferon
proteins are produced using the methods of the present invention. For
example, IFN-β variants are described in U.S. Pat. No. 6,531,122
"Interferon-β variants and conjugates," and U.S. Pat. No. 7,625,555,
both incorporated by reference herein. Conjugates include pegylated Type
1 interferons, e.g., the PEGylated agents shown in Table 4, and
interferons linked to non-peptide moieties.

[0090] The methods of the invention are expected to be useful for all Type
1 interferons, due to their structural similarities. Certain structurally
unrelated proteins, for example, human GCSF, have been found poor
candidates for producing using the methods of the present invention. When
GCSF was produced and extracted using methods as described herein, less
than 5% of the amount of GCSF protein in the insoluble fraction was
extracted as soluble protein (data not shown).

[0091] In general, with respect to an amino acid sequence, the term
"modification" includes substitutions, insertions, elongations,
deletions, and derivatizations alone or in combination. In some
embodiments, the peptides may include one or more modifications of a
"non-essential" amino acid residue. In this context, a "non-essential"
amino acid residue is a residue that can be altered, e.g., deleted or
substituted, in the novel amino acid sequence without abolishing or
substantially reducing the activity (e.g., the agonist activity) of the
peptide (e.g., the analog peptide). In some embodiments, the peptides may
include one or more modifications of an "essential" amino acid residue.
In this context, an "essential" amino acid residue is a residue that when
altered, e.g., deleted or substituted, in the novel amino acid sequence
the activity of the reference peptide is substantially reduced or
abolished. In such embodiments where an essential amino acid residue is
altered, the modified peptide may possess an activity of a Type 1
interferon of interest in the methods provided. The substitutions,
insertions and deletions may be at the N-terminal or C-terminal end, or
may be at internal portions of the protein. By way of example, the
protein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions,
both in a consecutive manner or spaced throughout the peptide molecule.
Alone or in combination with the substitutions, the peptide may include
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, again either in
consecutive manner or spaced throughout the peptide molecule. The
peptide, alone or in combination with the substitutions and/or
insertions, may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
deletions, again either in consecutive manner or spaced throughout the
peptide molecule. The peptide, alone or in combination with the
substitutions, insertions and/or deletions, may also include 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more amino acid additions.

[0092] Substitutions include conservative amino acid substitutions. A
"conservative amino acid substitution" is one in which the amino acid
residue is replaced with an amino acid residue having a similar side
chain, or physicochemical characteristics (e.g., electrostatic, hydrogen
bonding, isosteric, hydrophobic features). The amino acids may be
naturally occurring or normatural (unnatural). Families of amino acid
residues having similar side chains are known in the art. These families
include amino acids with basic side chains (e.g. lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic acid),
uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains
(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
tryptophan), β-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Substitutions may also include non-conservative
changes.

Methods for Selecting Optimal Extraction Conditions

[0093] In embodiments of the present invention, the results of the
statistical analysis as set forth in FIG. 4B are used to further optimize
extraction conditions within the ranges of parameter values provided.
High level soluble protein production of all Type 1 interferons is
expected to be observed when practicing the invention using any values
within the ranges set forth. Nonetheless, it is within the capacity of
one of skill in the art to utilize the tool represented by FIG. 4B to
optimize the extraction conditions to fit the need at hand.

Evaluation of Product

Protein Yield

[0094] Protein yield in the insoluble and soluble fractions as described
herein can be determined by methods known to those of skill in the art,
for example, by capillary gel electrophoresis (CGE), and Western blot
analysis.

[0095] Useful measures of protein yield include, e.g., the amount of
recombinant protein per culture volume (e.g., grams or milligrams of
protein/liter of culture), percent or fraction of recombinant protein
measured in the insoluble pellet obtained after lysis (e.g., amount of
recombinant protein in extract supernatant/amount of protein in insoluble
fraction), percent or fraction of active protein (e.g., amount of active
protein/amount protein used in the assay), percent or fraction of total
cell protein (tcp), amount of protein/cell, and percent dry biomass. In
embodiments, the measure of protein yield as described herein is based on
the amount of soluble protein or the amount of active protein, or both,
obtained.

[0096] In embodiments, the methods of the present invention can be used to
obtain an extracted recombinant protein yield of about 0.3 grams per
liter to about 10 grams per liter. In certain embodiments, the extracted
recombinant protein yield is about 0.3 grams per liter to about 1 gram
per liter, about 1 gram per liter to about 2 grams per liter, about 2
grams per liter to about 3 grams per liter, about 3 grams per liter to
about 4 grams per liter, about 4 grams per liter to about 5 grams per
liter, about 5 grams per liter to about 6 grams per liter, about 6 grams
per liter to about 7 grams per liter, about 7 grams per liter to about 8
grams per liter, about 8 grams per liter to about 9 grams per liter, or
about 9 grams per liter to about 10 grams per liter. In embodiments, the
extracted protein yield is about 1 gram per liter to about 3 grams per
liter, about 2 grams per liter to about 4 grams per liter, about 3 grams
per liter to about 5 grams per liter, about 4 grams per liter to about 6
grams per liter, about 5 grams per liter to about 7 grams per liter,
about 6 grams per liter to about 8 grams per liter, about 7 grams per
liter to about 9 grams per liter, or about 8 grams per liter to about 10
grams per liter. In embodiments, the extracted protein yield is about 0.5
grams per liter to about 4 grams per liter, 1 gram per liter to about 5
grams per liter, 2 grams per liter to about 6 grams per liter, about 3
grams per liter to about 7 grams per liter, about 4 grams per liter to
about 8 grams per liter, about 5 grams per liter to about 9 grams per
liter, or about 6 grams per liter to about 10 grams per liter. In
embodiments, the extracted protein yield is about 0.5 gram per liter to
about 5 grams per liter, about 1 grams per liter to about 6 grams per
liter, about 2 grams per liter to about 7 grams per liter, about 3 grams
per liter to about 8 grams per liter, about 4 grams per liter to about 9
grams per liter, or about 5 grams per liter to about 10 grams per liter.

[0097] In embodiments, the amount of recombinant interferon protein
detected in the extracted supernatant fraction is about 10% to about 95%
of the amount of the recombinant interferon protein detected in the
insoluble fraction. In embodiments, this amount is about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%, about 90%, or about 95%. In embodiments, this amount is about
10% to about 20%, 20% to about 50%, about 25% to about 50%, about 25% to
about 50%, about 25% to about 95%, about 30% to about 50%, about 30% to
about 40%, about 30% to about 60%, about 30% to about 70%, about 35% to
about 50%, about 35% to about 70%, about 35% to about 75%, about 35% to
about 95%, about 40% to about 50%, about 40% to about 95%, about 50% to
about 75%, about 50% to about 95%, or about 70% to about 95%.

[0098] The protein yield measured in the unextracted insoluble fraction is
typically higher than that in the extract supernatant, as material is
lost during the extraction procedure. Yields from fermentation cultures
are typically higher than those from smaller HTP cultures.

[0099] "Percent total cell protein" is the amount of protein or
polypeptide in the host cell as a percentage of aggregate cellular
protein. The determination of the percent total cell protein is well
known in the art.

[0100] In embodiments, the amount of interferon protein detected in the
extracted supernatant fraction produced is about 1% to about 75% of the
total cell protein. In certain embodiments, the recombinant protein
produced is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about
1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1%
to about 60%, about 1% to about 75%, about 2% to about 5%, about 2% to
about 10%, about 2% to about 20%, about 2% to about 30%, about 2% to
about 40%, about 2% to about 50%, about 2% to about 60%, about 2% to
about 75%, about 3% to about 5%, about 3% to about 10%, about 3% to about
20%, about 3% to about 30%, about 3% to about 40%, about 3% to about 50%,
about 3% to about 60%, about 3% to about 75%, about 4% to about 10%,
about 4% to about 20%, about 4% to about 30%, about 4% to about 40%,
about 4% to about 50%, about 4% to about 60%, about 4% to about 75%,
about 5% to about 10%, about 5% to about 20%, about 5% to about 30%,
about 5% to about 40%, about 5% to about 50%, about 5% to about 60%,
about 5% to about 75%, about 10% to about 20%, about 10% to about 30%,
about 10% to about 40%, about 10% to about 50%, about 10% to about 60%,
about 10% to about 75%, about 20% to about 30%, about 20% to about 40%,
about 20% to about 50%, about 20% to about 60%, about 20% to about 75%,
about 30% to about 40%, about 30% to about 50%, about 30% to about 60%,
about 30% to about 75%, about 40% to about 50%, about 40% to about 60%,
about 40% to about 75%, about 50% to about 60%, about 50% to about 75%,
about 60% to about 75%, or about 70% to about 75%, of the total cell
protein.

[0101] Solubility and Activity

[0102] The "solubility" and "activity" of a protein, though related
qualities, are generally determined by different means. Solubility of a
protein, particularly a hydrophobic protein, indicates that hydrophobic
amino acid residues are improperly located on the outside of the folded
protein. Protein activity, which can be evaluated using different
methods, e.g., as described below, is another indicator of proper protein
conformation. "Soluble, active, or both" as used herein, refers to
protein that is determined to be soluble, active, or both soluble and
active, by methods known to those of skill in the art.

[0103] Interferon Activity Assays

[0104] Assays for evaluating interferon protein activity are known in the
art and include binding activity assays that measure binding of
interferon to a standard ligand, e.g., a Blue sepharose column or a
specific antibody column.

[0105] Biological activity of interferons can be quantitated using known
assays, many of which are available commercially in kits. Most or all
interferon species have been shown to exert a similar spectrum of in
vitro biological activities in responsive cells, despite the existence of
different receptors for type I and type II IFN. Biological activities
induced by IFN include antiviral activity, which is mediated via cell
receptors and is dependent on the activation of signaling pathways, the
expression of specific gene products, and the development of antiviral
mechanisms. Sensitivity of cells to IFN-mediated antiviral activity is
variable, and depends on a number of factors including cell type,
expression of IFN receptors and downstream effector response elements,
effectiveness of antiviral mechanisms, and the type of virus used to
infect cells.

[0106] Biological activity assays include, e.g., cytopathic effect assays
(CPE), antiviral activity assays, assays that measure inhibition of cell
proliferation, regulation of functional cellular activities, regulation
of cellular differentiation and immunomodulation. Reporter gene assays
include the luciferase reporter cell-based assay described herein in the
Examples. In a reporter gene assay, the promoter region of an IFN
responsive gene is linked with a heterologous reporter gene, for example,
firefly luciferase or alkaline phosphatase, and transfected into an
IFN-sensitive cell line. Stably transfected cell lines exposed to IFN
increase expression of the reporter gene product in direct relation to
the dose of IFN, the readout being a measure of this product's enzymic
action. Many activity assay tools and kits are available commercially.
Biological assays for interferons are described, e.g., by Meager A,
"Biological assays for interferons," 1 Mar. 2002, J. Immunol. Methods
261(1-2):21-36, incorporated herein by reference.

[0107] In embodiments, activity is represented by the % active protein in
the extract supernatant as compared with the total amount assayed. This
is based on the amount of protein determined to be active by the assay
relative to the total amount of protein used in assay. In other
embodiments, activity is represented by the % activity level of the
protein compared to a standard, e.g., native protein. This is based on
the amount of active protein in supernatant extract sample relative to
the amount of active protein in a standard sample (where the same amount
of protein from each sample is used in assay).

[0108] In embodiments, about 40% to about 100% of the recombinant
interferon protein is determined to be active. In embodiments, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of
the recombinant interferon protein is determined to be active. In
embodiments, about 40% to about 50%, about 50% to about 60%, about 60% to
about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to
about 100%, about 50% to about 100%, about 60% to about 100%, about 70%
to about 100%, about 80% to about 100%, about 40% to about 90%, about 40%
to about 95%, about 50% to about 90%, about 50% to about 95%, about 50%
to about 100%, about 60% to about 90%, about 60% to about 95%, about 60%
to about 100%, about 70% to about 90%, about 70% to about 95%, about 70%
to about 100%, or about 70% to about 100% of the recombinant interferon
protein is determined to be active.

[0109] In other embodiments, about 75% to about 100% of the recombinant
interferon protein is determined to be active. In embodiments, about 75%
to about 80%, about 75% to about 85%, about 75% to about 90%, about 75%
to about 95%, about 80% to about 85%, about 80% to about 90%, about 80%
to about 95%, about 80% to about 100%, about 85% to about 90%, about 85%
to about 95%, about 85% to about 100%, about 90% to about 95%, about 90%
to about 100%, or about 95% to about 100% of the recombinant interferon
protein is determined to be active.

Expression Systems

[0110] Methods for expressing heterologous proteins, including useful
regulatory sequences (e.g., promoters, secretion leaders, and ribosome
binding sites), in Pseudomonas host cells, as well as host cells useful
in the methods of the present invention, are described, e.g., in U.S.
Pat. App. Pub. No. 2008/0269070 and U.S. patent application Ser. No.
12/610,207, both titled "Method for Rapidly Screening Microbial Hosts to
Identify Certain Strains with Improved Yield and/or Quality in the
Expression of Heterologous Proteins," U.S. Pat. App. Pub. No.
2006/0040352, "Expression of Mammalian Proteins in Pseudomonas
Fluorescens," and U.S. Pat. App. Pub. No. 2006/0110747, "Process for
Improved Protein Expression by Strain Engineering," all incorporated
herein by reference in their entirety. These publications also describe
bacterial host strains useful in practicing the methods of the invention,
that have been engineered to overexpress folding modulators or wherein
protease mutations have been introduced, in order to increase
heterologous protein expression. Sequence leaders are described in detail
in U.S. Patent App. Pub. No. 2008/0193974, "Bacterial leader sequences
for increased expression," and U.S. Pat. App. Pub. No. 2006/0008877,
"Expression systems with Sec-secretion," both incorporated herein by
reference in their entirety, as well as in U.S. patent application Ser.
No. 12/610,207.

Promoters

[0111] The promoters used in accordance with the present invention may be
constitutive promoters or regulated promoters. Common examples of useful
regulated promoters include those of the family derived from the lac
promoter (i.e. the lacZ promoter), especially the tac and trc promoters
described in U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16,
Ptac17, PtacII, PlacUV5, and the T7lac promoter. In one embodiment, the
promoter is not derived from the host cell organism. In certain
embodiments, the promoter is derived from an E. coli organism.

[0112] Inducible promoter sequences can be used to regulate expression of
interferons in accordance with the methods of the invention. In
embodiments, inducible promoters useful in the methods of the present
invention include those of the family derived from the lac promoter (i.e.
the lacZ promoter), especially the tac and trc promoters described in
U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16, Ptac17, PtacII,
PlacUV5, and the T7lac promoter. In one embodiment, the promoter is not
derived from the host cell organism. In certain embodiments, the promoter
is derived from an E. coli organism.

[0113] Common examples of non-lac-type promoters useful in expression
systems according to the present invention include, e.g., those listed in
Table 5.

[0114] See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) Manual of
Industrial Microbiology and Biotechnology (A. Demain & J. Davies, eds.)
pp. 460-74 (ASM Press, Washington, D.C.); H. Schweizer (2001) Current
Opinion in Biotechnology, 12:439-445; and R. Slater & R. Williams (2000
Molecular Biology and Biotechnology (J. Walker & R. Rapley, eds.) pp.
125-54 (The Royal Society of Chemistry, Cambridge, UK)). A promoter
having the nucleotide sequence of a promoter native to the selected
bacterial host cell also may be used to control expression of the
transgene encoding the target polypeptide, e.g, a Pseudomonas
anthranilate or benzoate operon promoter (Pant, Pben). Tandem promoters
may also be used in which more than one promoter is covalently attached
to another, whether the same or different in sequence, e.g., a Pant-Pben
tandem promoter (interpromoter hybrid) or a Plac-Plac tandem promoter, or
whether derived from the same or different organisms.

[0115] Regulated promoters utilize promoter regulatory proteins in order
to control transcription of the gene of which the promoter is a part.
Where a regulated promoter is used herein, a corresponding promoter
regulatory protein will also be part of an expression system according to
the present invention. Examples of promoter regulatory proteins include:
activator proteins, e.g., E. coli catabolite activator protein, MalT
protein; AraC family transcriptional activators; repressor proteins,
e.g., E. coli Lad proteins; and dual-function regulatory proteins, e.g.,
E. coli NagC protein. Many regulated-promoter/promoter-regulatory-protein
pairs are known in the art. In one embodiment, the expression construct
for the target protein(s) and the heterologous protein of interest are
under the control of the same regulatory element.

[0116] Promoter regulatory proteins interact with an effector compound,
i.e., a compound that reversibly or irreversibly associates with the
regulatory protein so as to enable the protein to either release or bind
to at least one DNA transcription regulatory region of the gene that is
under the control of the promoter, thereby permitting or blocking the
action of a transcriptase enzyme in initiating transcription of the gene.
Effector compounds are classified as either inducers or co-repressors,
and these compounds include native effector compounds and gratuitous
inducer compounds. Many
regulated-promoter/promoter-regulatory-protein/effector-compound trios
are known in the art. Although an effector compound can be used
throughout the cell culture or fermentation, in a preferred embodiment in
which a regulated promoter is used, after growth of a desired quantity or
density of host cell biomass, an appropriate effector compound is added
to the culture to directly or indirectly result in expression of the
desired gene(s) encoding the protein or polypeptide of interest.

[0117] In embodiments wherein a lac family promoter is utilized, a lad
gene can also be present in the system. The lad gene, which is normally a
constitutively expressed gene, encodes the Lac repressor protein Lad
protein, which binds to the lac operator of lac family promoters. Thus,
where a lac family promoter is utilized, the lad gene can also be
included and expressed in the expression system.

[0118] Promoter systems useful in Pseudomonas are described in the
literature, e.g., in U.S. Pat. App. Pub. No. 2008/0269070, also
referenced above.

Other Regulatory Elements

[0119] In embodiments, soluble proteins are present in either the
cytoplasm or periplasm of the cell during production. Secretion leaders
useful for targeting proteins are described elsewhere herein, and in U.S.
Pat. App. Pub. No. 2008/0193974, U.S. Pat. App. Pub. No. 2006/0008877,
and in U.S. patent application Ser. No. 12/610,207, referenced above.

[0120] An expression construct useful in practicing the methods of the
present invention can include, in addition to the protein coding
sequence, the following regulatory elements operably linked thereto: a
promoter, a ribosome binding site (RBS), a transcription terminator, and
translational start and stop signals. Useful RBSs can be obtained from
any of the species useful as host cells in expression systems according
to, e.g., U.S. Pat. App. Pub. No. 2008/0269070 and U.S. patent
application Ser. No. 12/610,207. Many specific and a variety of consensus
RBSs are known, e.g., those described in and referenced by D. Frishman et
al., Gene 234(2):257-65 (8 Jul. 1999); and B. E. Suzek et al.,
Bioinformatics 17(12):1123-30 (December 2001). In addition, either native
or synthetic RBSs may be used, e.g., those described in: EP 0207459
(synthetic RBSs); O. Ikehata et al., Eur. J. Biochem. 181(3):563-70
(1989) (native RBS sequence of AAGGAAG). Further examples of methods,
vectors, and translation and transcription elements, and other elements
useful in the present invention are described in, e.g.: U.S. Pat. No.
5,055,294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroy et al.; U.S.
Pat. No. 5,281,532 to Rammler et al.; U.S. Pat. Nos. 4,695,455 and
4,861,595 to Barnes et al.; U.S. Pat. No. 4,755,465 to Gray et al.; and
U.S. Pat. No. 5,169,760 to Wilcox.

Host Strains

[0121] Bacterial hosts, including Pseudomonas, and closely related
bacterial organisms are contemplated for use in practicing the methods of
the invention. In certain embodiments, the Pseudomonas host cell is
Pseudomonas fluorescens. The host cell can also be an E. coli cell.

[0122] Pseudomonas and closely related bacteria are generally part of the
group defined as "Gram(-) Proteobacteria Subgroup 1" or "Gram-Negative
Aerobic Rods and Cocci" (Buchanan and Gibbons (eds.) (1974) Bergey's
Manual of Determinative Bacteriology, pp. 217-289). Pseudomonas host
strains are described in the literature, e.g., in U.S. Pat. App. Pub. No.
2006/0040352, cited above.

[0126] Methods for optimizing codons to improve expression in bacterial
hosts are known in the art and described in the literature. For example,
optimization of codons for expression in a Pseudomonas host strain is
described, e.g., in U.S. Pat. App. Pub. No. 2007/0292918, "Codon
Optimization Method," incorporated herein by reference in its entirety.

[0128] The expression system according to the present invention can be
cultured in any fermentation format. For example, batch, fed-batch,
semi-continuous, and continuous fermentation modes may be employed
herein.

[0129] In embodiments, the fermentation medium may be selected from among
rich media, minimal media, and mineral salts media. In other embodiments
either a minimal medium or a mineral salts medium is selected. In certain
embodiments, a mineral salts medium is selected.

[0130] Mineral salts media consists of mineral salts and a carbon source
such as, e.g., glucose, sucrose, or glycerol. Examples of mineral salts
media include, e.g., M9 medium, Pseudomonas medium (ATCC 179), and Davis
and Mingioli medium (see, B D Davis & E S Mingioli (1950) J. Bact.
60:17-28). The mineral salts used to make mineral salts media include
those selected from among, e.g., potassium phosphates, ammonium sulfate
or chloride, magnesium sulfate or chloride, and trace minerals such as
calcium chloride, borate, and sulfates of iron, copper, manganese, and
zinc. Typically, no organic nitrogen source, such as peptone, tryptone,
amino acids, or a yeast extract, is included in a mineral salts medium.
Instead, an inorganic nitrogen source is used and this may be selected
from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia. A
mineral salts medium will typically contain glucose or glycerol as the
carbon source. In comparison to mineral salts media, minimal media can
also contain mineral salts and a carbon source, but can be supplemented
with, e.g., low levels of amino acids, vitamins, peptones, or other
ingredients, though these are added at very minimal levels. Media can be
prepared using the methods described in the art, e.g., in U.S. Pat. App.
Pub. No. 2006/0040352, referenced and incorporated by reference above.
Details of cultivation procedures and mineral salts media useful in the
methods of the present invention are described by Riesenberg, D et al.,
1991, "High cell density cultivation of Escherichia coli at controlled
specific growth rate," J. Biotechnol. 20 (1):17-27.

[0131] Fermentation may be performed at any scale. The expression systems
according to the present invention are useful for recombinant protein
expression at any scale. Thus, e.g., microliter-scale, centiliter scale,
and deciliter scale fermentation volumes may be used, and 1 Liter scale
and larger fermentation volumes can be used.

[0132] In embodiments, the fermentation volume is at or above about 1
Liter. In embodiments, the fermentation volume is about 1 liter to about
100 liters. In embodiments, the fermentation volume is about 1 liter,
about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6
liters, about 7 liters, about 8 liters, about 9 liters, or about 10
liters. In embodiments, the fermentation volume is about 1 liter to about
5 liters, about 1 liter to about 10 liters, about 1 liter to about 25
liters, about 1 liter to about 50 liters, about 1 liter to about 75
liters, about 10 liters to about 25 liters, about 25 liters to about 50
liters, or about 50 liters to about 100 liters In other embodiments, the
fermentation volume is at or above 5 Liters, 10 Liters, 15 Liters, 20
Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500
Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or
50,000 Liters.

[0133] While preferred embodiments of the present invention have been
shown and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only. Numerous
variations, changes, and substitutions will now occur to those skilled in
the art without departing from the invention. It should be understood
that various alternatives to the embodiments of the invention described
herein may be employed in practicing the invention. It is intended that
the following claims define the scope of the invention and that methods
and structures within the scope of these claims and their equivalents be
covered thereby.

EXAMPLES

Example 1

Production of rIFN-β from High Throughput Expression Samples

[0134] In the following experiment, IFN-β C17S expression strains
were constructed, and the amount of IFN-β in the insoluble fraction
obtained for each was quantitated. Based on the resulting data, certain
strains were selected for use in optimizing the non-denaturing extraction
process of the present invention.

[0137] Plasmids were constructed which carry the codon-optimized
IFN-β fused to nineteen P. fluorescens secretion leaders. The
secretion leaders were included to target the protein to the periplasm
where it may be recovered in the properly folded and active form. In
addition, one plasmid was constructed which carries the codon-optimized
IFN-β designed for cytoplasmic expression.

[0138] Expression of IFN-β was driven from the Ptac promoter and
translation initiated from either a high (Hi) or medium (Med) activity
ribosome binding site (RBS). The resulting 20 plasmids were transformed
into 30 P. fluorescens host strains (16 protease deletion strains, 13
folding modulator overexpression strains and 1 wild type strain) to
produce 600 expression strains (see Tables 6 and 7). Folding modulators,
when present, were encoded on a second plasmid and expression driven by a
mannitol inducible promoter.

[0139] The thirty host strains carrying each of 20 IFN-β expression
plasmids (600 expression hosts in total) were grown in triplicate in
96-well plates. Samples harvested 24 hours after induction were used for
analysis.

[0141] Ten microliters of seed culture were transferred into triplicate
wells of 96-well deep well plates, each well containing 500 microliters
of HTP-YE medium, and incubated as before for 24 hours.
Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to each well
for a final concentration of 0.3 mM to induce the expression of
IFN-β. For growth of small cultures in HTP microwells, a specific
culture pH is not tightly controlled and the cell density can differ
slightly from well to well. Mannitol (Sigma, M1902) was added to each
well at a final concentration of 1% to induce the expression of folding
modulators in folding modulator over-expressing strains, and the
temperature was reduced to 25° C. Twenty four hours after
induction, cultures were collected for analysis. For OD normalization,
cells were mixed with sterile 1×PBS to obtain a final OD600=20 in a
final volume of 400 microliters using the Biomek liquid handling station
(Beckman Coulter). Samples were collected in cluster tube racks.

Sample Preparation and SDS-CGE Analysis

[0142] Soluble fractions (supernatants obtained after centrifugation of
lysates) and insoluble fractions (pellets obtained after centrifugation
of lysates) were prepared by sonicating the OD-normalized cultures,
followed by centrifugation. Frozen, normalized culture broth (400
microliters) was thawed and sonicated for 3.5 minutes. The lysates were
centrifuged at 20,800×g for 20 minutes (4° C.) and the
soluble fractions removed using manual or automated liquid handling. The
insoluble fractions were frozen and then thawed for re-centrifugation at
20,080×g for 20 minutes at 4° C., to remove residual
supernatant. The insoluble fractions were then resuspended in 400 μL
of 1× phosphate buffered saline (PBS), pH 7.4. Further dilutions of
soluble and insoluble fractions for SDS-CGE analysis were performed in
1× phosphate buffered saline (PBS), pH 7.4. Soluble and insoluble
fractions were prepared for analysis by SDS capillary gel electrophoresis
(CGE) (Caliper Life Sciences, Protein Express LabChip Kit, Part 760301),
in the presence of dithiothreitol (DTT).

[0143] Normalized soluble and insoluble fractions from each well of the
600 strains expressing IFN-β were analyzed by reducing SDS-CGE
analysis in one replicate for the soluble fractions and insoluble
fractions. No IFN-β signal was detected in the soluble fractions.
IFN-β signal varied from no signal to greater than 400 mg/L in the
insoluble fractions. Only five of the twenty plasmids tested showed
measurable signal of IFN-β in the insoluble fractions of all thirty
host strains: p530-001, p530-007, p530-011, p530-018 and p530-020. Valley
to valley integration of IFN-β signal using Caliper LabChipGX
software was performed in all 150 strains consisting of the five plasmids
listed above in the thirty host strains, and data were used to calculate
volumetric yields. Volumetric yields of the 150 strains analyzed ranged
from 2 to 482 mg/L. Strains carrying p530-020 attained consistently
higher yields of IFN-β in the insoluble fraction than other
expression strains; however, the protein migrated higher than expected on
SDS-CGE, indicating that the secretion leader was not cleaved. High
yields were also observed with 2 host strains carrying p530-001. No
significant difference in IFN-β in the insoluble fraction was
observed among the 30 strains except potentially in one strain, DC441, a
Ion hslUV protease deletion strain, which showed somewhat higher yields
than the other 29 strains.

[0144] A subset of 17 top expression strains (Table 8), excluding strains
containing plasmid p530-020, was selected for further analyses. The
expression strains containing plasmid p530-020 were excluded from further
consideration in this experiment due to the potentially unprocessed
leader. SDS-CGE analysis was performed on the soluble and insoluble
fractions for these strains. Quantification of the SDS-CGE output is
shown in Table 8. IFN-β protein concentration ranged from 102 to
greater than 482 mg/L. Based upon insoluble yield and processing of
either the periplasmic leader sequence or the N-terminal Met from
IFN-β, strains were chosen to proceed to fermentation assessment.

[0146] HTP expression plate cultures of Pseudomonas fluorescens strains
PS530-001 overexpressing cytoplasmic IFN-β 1b and 530-220,
overexpressing secreted IFN-β 1b (described in Example 1), were
sonicated and centrifuged to obtain an insoluble fraction and a soluble
fraction. The pellets were resuspended in extraction buffer 1×PBS,
pH 7.4 or sodium acetate at pH 4.5. Each buffer was tested either with or
without Zwittergent 3-14 detergent at 1% (w/v). Each of the four
combinations of buffer and detergent was incubated for 1-2 hours at room
temperature or overnight at 4° C. with shaking. After extraction,
each sample was centrifuged for 20 minutes at 20,080×g at 4°
C. to produce a second insoluble pellet fraction (extract pellet) and a
second soluble supernatant fraction (extract supernatant). The first
insoluble fraction and first soluble fraction, and the extract pellet
fraction and extract supernatant fraction, were analyzed by SDS-CGE. The
results are shown in FIGS. 1A and 1B. As seen in Lane 7, the extraction
condition including PBS buffer and Zwittergent 3-14 yielded soluble
IFN-β.

Example 3

Optimization of Conditions for Extraction

[0147] Insoluble fractions from fermentation cultures were extracted under
conditions comprising different detergents.

[0148] Frozen cell paste from a 1 L fermentation (grown at 32° C.,
pH 6.5, and induced using 0.2 mM IPTG at an OD575 of 100) of strain
PS530-001, overexpressing recombinant IFN-β 1b, was resuspended in
lysis buffer containing 20 mM sodium phosphate (JT Baker), pH 7.4 to a
final solids concentration of 20% (w/v). The well-mixed cell slurry was
lysed with two passes at 38 kpsi through a Constant cell disruptor
(Constant Systems, Inc.). The lysate was split in half, and spun by
centrifugation at 15,000×g for 30 minutes at 4° C. (Beckman
Coulter, PN# J-20, XPF). The pellets (containing IFN-β and cell
debris) were resuspended and each was washed in either Buffer A (20 mM
sodium phosphate, pH 7.4) or Buffer B (20 mM sodium acetate, pH 4.0).
Samples were spun by centrifugation under the same conditions described
for the first spin, supernatants were removed, and the pellets were again
resuspended in either Buffer A or B at 20% solid concentration. For each
buffer, twenty aliquots of 1 mL each were placed in 1.5 mL conical tubes.
Detergent stock solutions were added to the conical tubes at different
concentrations. All tubes were incubated at room temperature for 1 hour
or overnight (18 hours) at 4° C. with continuous mixing. After
extraction, the solutions were centrifuged and the extract supernatant
fractions were analyzed for protein concentration by SDS-CGE. FIG. 2
provides a flow chart showing how the sample preparation and extraction
were carried out.

[0149] Of the detergents tested, Zwittergent 3-14 and N-lauroylsarcosine
(NLS), were found to give the best yields regardless of buffer and
incubation time (Table 9). However, the product extracted using NLS was
not active, as determined by its inability to bind to either a Blue
Sepharose affinity column or a cation exchange column (SP HP) (data not
shown). The product extracted using Zwittergent 3-14 was determined to be
active.

[0150] Using similar methods, Zwittergent analogs were evaluated for their
extraction efficiency. The results are shown in Table 10. The best yields
were observed with Zwittergent 3-14. Zwittergent 3-12, Zwittergent 3-10,
and Zwittergent 3-08 were also effective.

[0151] To efficiently solubilize proteins, the detergent concentration
typically needs to be above its CMC value. The CMC of Zwittergent 3-14 is
about 0.01% w/v. Extraction conditions including sodium phosphate buffer
at pH 7.4 with increasing concentrations of Zwittergent 3-14 were
evaluated. The cell paste used was obtained by growing strain PS530-001
at 32° C., pH 6.5, and induced using 0.2 mM IPTG at an OD575
of 100. The results in Table 11 show that use of Zwittergent 3-14 at 1%
(w/v) concentration resulted in the highest extraction yield.

[0152] As shown in Table 11, extraction conditions including Zwittergent
3-14 at 1% (w/v) concentration in sodium phosphate buffer at pH 7.4
yielded 21% of the IFN-β 1b detected in the original insoluble
fraction. Further optimization was conducted.

[0153] High concentration (e.g., 6 to 8 M) of some chaotropic reagents
like urea and guanidinium hydrochloride commonly have been used as a
strong denaturant for solubilization of inclusion bodies. Chaotropes such
as urea can increase the detergent critical micelle concentration (CMC)
and may potentially improve the extraction efficiency. Low concentrations
of urea (up to 2 M) were evaluated in the extraction conditions. Salts,
e.g., NaCl, can also affect detergent CMC. Varying Zwittergent 3-14
concentrations were evaluated due to the potential interplay between
detergent CMC and the presence of chaotrophic reagents and salts. The
concentration of insoluble inclusion solids in the extraction conditions
was also varied. Lower solids concentration than the 20% (w/v) previously
used were evaluated.

[0154] In summary, the effect of varying the following parameters on
extraction efficiency was tested.

[0155] Sodium Chloride: 150-1850 mM

[0156] Urea: 0-2 M

[0157] Zwittergent 3-14: 0.1-1.0% w/v

[0158] Solids: 5-20% w/v

[0159] pH: 6.5-8.5

[0160] The flow chart in FIG. 3 describes the preparation and extraction
of the first insoluble pellet fraction for this optimization study. Table
12 shows the result of the study. FIGS. 4A and B summarize the results
and significance of the effect of each parameter on the extraction yield.
For optimization of extraction of interferon 0 from the insoluble
fraction, a two-level five-factor half-fractional factorial experimental
design was used. JMP software (SAS Institute, Cary, N.C.) was used for
experimental design and analysis. The software estimates the effect of
individual factors as well as interactions on experimental output (amount
of interferon extracted).

[0161] Based on the above data, an optimized extraction condition was
selected for experiments described hereinafter: 1% (w/v) Zwittergent
3-14, 2 M Urea, 2 M NaCl, Solids 5% w/v, buffer pH 7.5 to 8.5. Using
these optimized conditions, the observed extraction yield (in the extract
supernatant) was found to be consistently 90% or above (i.e., 90% or more
of the amount of recombinant protein measured in the insoluble fraction).

[0163] Fermentation cultures were grown in 2 liter fermentors containing a
mineral salts medium (as described herein and also by, e.g., Riesenberg,
D., et al., 1991). Culture conditions were maintained at 32° C.
and pH 6.5 through the addition of aqueous ammonia. Dissolved oxygen was
maintained in excess through increases in agitation and flow of sparged
air and oxygen into the fermentor. Glycerol was delivered to the culture
throughout the fermentation to maintain excess levels. These conditions
were maintained until the target culture optical density (A575) for
induction was reached, at which time IPTG was added to initiate
IFN-β production. The optical density at induction, the
concentration of IPTG, pH and temperature were all varied to determine
optimal conditions for expression. After 24 hours, the culture from each
fermentor was harvested by centrifugation and the cell pellet frozen at
-80° C.

[0164] Fermentation cultures were induced at 100 OD575 using 0.2 mM
IPTG, at pH 6.5 and a temperature of 32° C. Replicate
fermentations resulted in IFN-β production at 7.5, 8.4 and 7.9 g/L
as determined by SDS-CGE of the initial insoluble fraction (FIG. 5). When
these insoluble fractions were subjected to extraction (under conditions
including 1% (w/v) Zwittergent 3-14, 2 M Urea, 2 M NaCl, Solids 5% w/v,
and buffer pH 8.2), solubilized IFN-β were observed in the extract
supernatant at 2.2, 2.4, and 2.6 g/L. This represents an average
extraction yield of 31%.

[0165] Increasing the induction OD to 120 to 160, and decreasing the
fermentation pH to 5.7 to 6.25, increased IFN-β titers in the
initial insoluble fraction to 8.8-9.2 g/L (FIG. 6). Extraction of these
cell pellets (using the same extraction conditions as for the experiment
shown in FIG. 5) resulted in 3.1 to 4.0 g/L of IFN-β in the
extracted supernatant fraction, an average extraction yield of 39% (Table
13).

[0166] Broth from fermentation of Pseudomonas fluorescens strain PS530-001
(1 L fermentation at 32° C., pH 6.0, induced at OD575 of 100
using 0.2 mM IPTG) was centrifuged and the supernatant discarded. The
cell paste was resuspended in 20 mM Tris, pH 8.2 (in a ratio of 1:4) and
lysed by passing through Microfluidics Microfluidizer M110Y at 15,000
psi. The lysate was centrifuged and the soluble fraction discarded. The
insoluble fraction was mixed with extraction buffer (20 mM Tris, 2 M
NaCl, 2 M urea, 1% Zwittergent 3-14, pH 8.2) at room temperature for 1
hour and centrifuged to produce an extract supernatant fraction and an
extract pellet fraction. The extraction yield of IFN-β (IFN-β
in extract supernatant fraction/IFN-β in the initial insoluble
fraction) was close to 100% (>99%) based on SDS-CGE analysis (data not
shown).

[0167] The extract supernatant was filtered and loaded on a 5 mL GE
Healthcare Blue Sepharose column equilibrated with 20 mM Tris, 2 M NaCl,
pH 8.2. The column was washed with the same buffer and the IFN-β
eluted with 20 mM Tris, 2 M NaCl, 50% propylene glycol, pH 8.2. The
protein in the elution pool was analyzed by SDS-CGE and found to be more
than 98% pure IFN-β. Aliquots of the elution pool were exchanged
into Buffers C (5 mM glycine pH 3.0) and D (5 mM aspartic acid, 9%
trehalose, pH 4.0).

[0168] The exchanged samples were analyzed by SDS-CGE as well as with a
cell-based assay (PBL Interferon Source, #51100-1). The cell-based assay
uses a human cell line (PIL5) sensitized with IFN-type 1 receptor.
IFN-β binds to the receptor, which sends a signal via the Jak1/STAT1
signal transduction pathway, activating ISG15-luciferase transcription
via the Interferon Sensitive Response Element (ISRE). Cell-based assay
kit instructions were followed as per manufacturer's protocol (51100
rev01). The signal was read using conventional plate readers with
luminescence detection. Table 14 summarizes the SDS-CGE and cell-based
assay results, which indicate that the IFN-β in the samples was
fully active.

[0170] Plasmids expressing either protein were constructed and transformed
into different host strains. Expression strains were tested for their
ability to express recombinant protein using HTP analysis, as described
with regard to IFN-β herein. A subset of the expression strains are
selected for fermentation studies.

[0171] The selected strains were grown and induced according to the
present invention. The cells were centrifuged, lysed, and centrifuged
again as described herein for IFN-β. The resulting insoluble
fraction and first soluble fraction were extracted using extraction
conditions described herein. The resulting IFN-α 2a and IFN-α
2b extract supernatants were quantitated using SDS-CGE (data not shown).

Example 7

Extraction of IFN-α 2a and 2b from High Throughput Expression
Material

[0172] The first insoluble fraction obtained as described in Example 6 is
extracted using the extraction conditions of the present invention.
IFN-α 2a and 2b in the resulting second soluble fractions are
evaluated by CGE and bioactivity assay.

Example 8

Production of IFN-α 2a and 2b from Large Scale Fermentation

[0173] IFN-α 2a and 2b expressing strains selected by HTP analysis
are grown in 2 liter fermentors using optimized fermentation conditions
of the present invention, e.g., as described in Example 4. The first
insoluble fraction is extracted using the methods of the present
invention, e.g., as described in Example 4. The IFN-α 2a and 2b
protein present in the first insoluble and second soluble fractions are
evaluated by CGE and compared.

Example 9

Analysis of IFN-α 2a and 2b Extraction Product

[0174] The extraction product obtained in Example 8 is analyzed for
IFN-α 2a and 2b bioactivity.